KR20190044059A - A novel alkaline stable immunoglobulin binding protein - Google Patents

Abstract

The present invention relates to immunoglobulin (Ig) binding proteins comprising at least one Ig binding domain having an amino acid selected from the group consisting of at least 1I, 11A, 11E, 11I, 35R, 35I and 42L. The invention further relates to an affinity matrix comprising an Ig binding protein of the invention. The present invention also relates to the use of said Ig binding protein or affinity matrix for affinity purification of immunoglobulin and to affinity purification methods using the Ig binding protein of the present invention.

Description

A novel alkaline stable immunoglobulin binding protein

The present invention relates to an alkaline stable immunoglobulin (Ig) binding protein comprising at least one Ig binding domain having an amino acid selected from the group consisting of at least 11, 11A, 11E, 11I, 35R, . The invention further relates to affinity matrices comprising an alkali stable Ig binding protein of the invention. The present invention also relates to the use of said Ig binding protein or affinity matrix for affinity purification of immunoglobulin and to affinity purification methods using the Ig binding protein of the present invention.

Many biotechnological and pharmaceutical applications require the removal of contaminants from samples containing antibodies. Procedures established for the capture and purification of antibodies include the use of staphylococcus as an optional ligand for immunoglobulins Is an affinity chromatography using bacterial cell surface protein A from Staphylococcus aureus (see, for example, review Huse et al., J. Biochem. Biophys. Methods 51, 2002: 217-231). Wild type protein A binds to the Fc region of IgG molecules with high affinity and selectivity, and is also stable at high temperature and a wide range of pH values. Variants of protein A with improved properties such as alkaline stability are available for antibody purification and a variety of chromatographic matrices including protein A ligands are commercially available. However, specifically, the wild-type protein A-based chromatography matrix showed a loss of binding ability to immunoglobulin after exposure to alkaline conditions.

Most large-scale manufacturing processes for antibody or Fc-containing fusion proteins use protein A for affinity purification. However, due to the limitations of protein A application in affinity chromatography, there is a need for providing novel Ig binding proteins with improved properties that specifically bind to immunoglobulins to facilitate affinity purification of immunoglobulins ≪ / RTI > In order to make full use of the value of the chromatography matrix comprising the Ig binding protein, it is desirable to use multiple affinity ligand matrices. Between chromatographic cycles, thorough cleaning procedures are required for sterilization and removal of residual contaminants on the matrix. In this procedure it is common practice to apply an alkaline solution with a high concentration of NaOH to the affinity ligand matrix. The wild-type protein A domain does not tolerate these harsh alkaline conditions for extended periods of time and rapidly abolishes binding to immunoglobulins. Thus, there is a need in the art for obtaining a novel alkaline-stable protein capable of binding to immunoglobulins.

The present invention provides an alkali stable immunoglobulin binding protein that is particularly well suited for affinity purification of immunoglobulins and which overcomes the disadvantages of the prior art. In particular, a significant advantage of the alkali stable Ig binding proteins of the present invention lies in its improved stability at higher pH compared to the parental protein.

The above summary does not necessarily describe all the problems that are solved by the present invention.

The first aspect of the present invention is to provide an Ig binding protein suitable for affinity purification. Which is achieved by an alkali stable Ig binding protein comprising at least one Ig binding domain, wherein said at least one Ig binding domain comprises an amino acid substitution at position 1 or an equivalent position to isoleucine at position 11 or a corresponding position Alanine, glutamic acid, or isoleucine, amino acid substitution at position 35 or its corresponding position to arginine or isoleucine, and amino acid substitution at position 42 or its corresponding position to leucine, 2, 3, or 4 substitutions of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the invention comprises an Ig binding protein and the at least one Ig binding domain comprises the consensus amino acid sequence of SEQ ID NO: 52.

In a second aspect, the invention relates to an affinity separation matrix comprising an alkali stable Ig binding protein of the first aspect.

In a third aspect, the invention relates to the use of an alkali stable Ig binding protein of the first aspect or an affinity separation matrix of the second aspect for affinity purification of an immunoglobulin or protein comprising an Fc portion of an immunoglobulin.

In a fourth aspect, the present invention relates to a method of affinity purification of an immunoglobulin or protein comprising an Fc portion of an immunoglobulin, said method comprising the steps of: (a) providing a liquid containing an immunoglobulin; (b) providing an affinity separation matrix comprising an immobilized alkali stable Ig binding protein of a first aspect coupled to an affinity separation matrix; (c) contacting the liquid with the affinity separation matrix, wherein the immunoglobulin binds to the immobilized Ig binding protein; And (d) eluting the immunoglobulin from the matrix, thereby obtaining an eluate containing the immunoglobulin.

The summary of the invention does not necessarily describe all features of the invention. Other embodiments will become apparent from a review of the following detailed description.

Figure 1. Amino acid sequence of an alkali stable Ig binding domain. Positions 1, 11, 35, and 42 are shown in gray. The numbers in the top row represent the corresponding amino acid positions in the Ig binding domain.
Fig. 1A. Amino acid sequence of an artificial alkaline stable Ig binding domain.
Fig. 1B. Consensus amino acid sequence of an alkaline stable artificial Ig binding domain (SEQ ID NO: 52).
Figure 2. Analysis of alkaline stability of point mutant variants of maternal IB14. The remaining activity (%) of Ig binding after 6 hours of continuous 0.5 M NaOH treatment of mutants with point mutations at position 1, 11, 35, or 42 was compared to maternal IB14.
Figure 3. Analysis of the alkali stability of different variants of the parent IB14 with substitutions at positions 1, 11, and 35, and optionally at positions 28 and 42. (%) (Black bars) of Ig binding after 6 hours of continuous 0.5 M NaOH treatment of the combination of substitutions at positions 1, 11, 28, 35, and / or 42 compared to matrix IB14 (light gray bars) Respectively. cs14-1 represents SEQ ID NO: 18 (1I / 11A / 35R), cs14-2 represents (1I / 11A / 35R / 42L) SEQ ID NO: 19, cs14-3 represents SEQ ID NO: 20 / 28N / 35R / 42L).
Figure 4. Analysis of the alkali stability of different variants of the parent IB27 with positions 1, 11, and 35, and optionally combinations of 3 or 4 substitutions at 42. The remaining activity (%) of Ig binding (black bars) was compared to maternal IB27 (light gray bars) after 6 hours of continuous 0.5 M NaOH treatment of variant Ig binding proteins. cs27-1 represents SEQ ID NO: 29 (1I / 11A / 35R), and cs27-2 represents SEQ ID NO: 30 (1I / 11A / 35R / 42L).
Figure 5. Ig binding activity of the Ig binding domain after alkaline treatment.
Analysis of the alkali stability of the different Ig binding domains on the epoxy resin after 6 hours of 0.5 M NaOH treatment. 1I, 11A, 35R, and 42L. (SEQ ID NO: 42), cs74h2 (SEQ ID NO: 43), cs47h3 (SEQ ID NO: 44), and cs47h4 (SEQ ID NO: 45), cs25 -2 (SEQ ID NO: 26); Matrix domain: IB14.

Justice

Before disclosing the invention in detail below, it should be understood that the invention is not limited to the particular methodology, protocols and reagents disclosed herein, as they may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is to be limited only by the terms of the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Preferably, as used herein, the term "A multilingual glossary of biotechnological terms" (IUPAC Recommendations), Leuenberger, H. G. W., Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word " comprise ", and variations such as " comprises " and " comprising " Integers or steps or members, integers, or groups of steps, but does not imply the exclusion of any other element, integer or step or group of elements, integers, or groups of steps.

As used in the specification and appended claims, the singular forms " a, " an, and " the " are used interchangeably and, unless the context clearly requires otherwise, . Also, as used herein, the term " and / or " refers to and does not include combinations of any of the listed items and any and all possible combinations thereof, as well as alternatives ("or").

As used herein, the term " about " encompasses not only the amount explicitly quoted, but also a deviation of +/- 10% therefrom. More preferably, the deviation of 5% is encompassed by the term " about ".

Certain documents (eg, patents, patent applications, scientific literature, manufacturer's specifications, guidelines, GenBank accession number sequence submissions, etc.) are cited throughout the text of this specification. And should not be interpreted as admitting herein that the present invention is not entitled to antedate such disclosure by a prior invention. Some publications cited herein are & quot ; included by reference & quot ;. In the event of a conflict between the definitions or teachings of such incorporated references and the definitions or teachings cited herein, the description herein shall control.

All of the sequences referred to herein, along with their entire contents and disclosure, are set out in the accompanying Sequence Listing which is a part of this specification.

The term "immunoglobulin-binding protein" or "Ig binding protein" or "immunoglobulin (Ig) binding protein" in the context of the present invention is used to describe a protein that can specifically bind to the Fc region of an immunoglobulin do. Due to this specific binding to the Fc region, the " Ig binding protein " of the present invention includes whole immunoglobulin, an immunoglobulin fragment comprising the Fc region, a fusion protein comprising the Fc region of the immunoglobulin, and an Fc region of the immunoglobulin May be conjugated to an included conjugate. Although the Ig binding proteins of the present invention herein exhibit specific binding to the Fc region of an immunoglobulin, it is excluded that the Ig binding protein can be further bound with reduced affinity for other regions of the immunoglobulin, such as the Fab region It is not.

In a preferred embodiment of the invention, the Ig binding protein comprises at least one alkaline stable Ig binding domain.

The term " dissociation constant " or " K _D " defines a specific binding affinity. As used herein, the term "K _D " (commonly referred to as "mol / L", sometimes abbreviated as "M") refers to the dissociation equilibrium constant of a particular interaction between a first protein and a second protein . In the context of the present invention, the term K _D is specifically used to describe the binding affinity between an Ig binding protein and an immunoglobulin.

An Ig binding protein of the invention is considered to bind to an immunoglobulin, which is at least 1 [mu] M or less, or preferably 100 nM or less, more preferably 50 nM or less, more preferably 10 nM or less And a dissociation constant K _D for immunoglobulin.

The term " bond " according to the invention is preferably concerned with specific bonding. By " specific binding " is meant that the Ig binding protein of the invention binds more specifically to immunoglobulin compared to binding to other non-immunoglobulin targets.

The term " immunoglobulin " or " Ig ", as used interchangeably herein, includes proteins having a four-polypeptide chain structure consisting of two heavy chains and two light chains with the ability to specifically bind to an antigen . Additionally, fragments or variants thereof are also encompassed by the term " immunoglobulin ". It is understood herein that Ig fragments are intact or contain less amino acid residues than the entire Ig. The term also encompasses embodiments such as chimeras (human constant domains, non-human variable domains), single chain and humanized (human antibodies other than non-human CDR) immunoglobulins.

"Immunoglobulin" as understood herein includes, but is not limited to, mammalian IgG such as human IgG ₁ , human IgG ₂ , human IgG ₄ , mouse IgG ₁ , mouse IgG ₂ A, mouse IgG ₂ IgG ₁ , IgG ₂ C, goat IgG ₁ , goat IgG ₂ , small IgG ₂ , guinea pig IgG, rabbit IgG; Human IgM, human IgA; And an immunoglobulin fragment comprising an Fc region, a fusion protein comprising an Fc region of an immunoglobulin, and a conjugate comprising an Fc region of an immunoglobulin. In particular, the protein A domain, and an artificial Ig binding protein naturally occurring in the present invention do not bind to human IgG _3.

The terms " protein " and " polypeptide " refer to any linear molecular chain of two or more amino acids joined by peptide bonds and do not represent a product of a particular length. Thus, any other term used to denote a chain of two or more amino acids is included within the definition of " polypeptide ", and the term " polypeptide " Alternatively, or in an interchangeable manner. The term " polypeptide " also encompasses polypeptides, including but not limited to glycosylation, acetylation, phosphorylation, amidation, proteolytic cleavage, modification by non-naturally occurring amino acids and similar variations well known in the art Translation-post-transformation. Thus, Ig binding proteins comprising two or more protein domains also fall within the definition of the term " protein " or " polypeptide ".

The term "alkali stable" or "alkali stable" or "corrosion stable" or "corrosion stability" (abbreviated herein as "cs") is intended to encompass the present invention Ig binding protein. One of ordinary skill in the art will recognize that the Ig binding protein may be incubated with a solution of sodium hydroxide as described in the Examples, followed by a routine experiment known to one of ordinary skill in the art, for example by immunoassay The binding activity can be tested to easily test for alkali stability.

The matrix comprising the Ig binding protein of the present invention as well as the Ig binding protein of the present invention exhibits " increased " or " improved " alkali stability because the molecules and the matrix comprising the Ig binding protein are extended Means that it is stable under alkaline conditions for a period of time, that is, does not lose its ability to bind immunoglobulin or lose its ability to bind immunoglobulin to a lesser extent than the parent protein.

The term " binding activity " refers to the ability of the Ig binding proteins of the invention to bind to immunoglobulins. For example, the binding activity can be determined before and / or after the alkali treatment. The binding activity can be determined for Ig binding proteins or for Ig binding proteins that are coupled to the matrix, i. E., For immobilized binding proteins. The term " artificial " refers to an object that does not occur naturally, i.e. the term refers to an object produced or modified by a human. For example, a polypeptide or polynucleotide sequence generated by a human (e.g., by genetic engineering, shuffling, or chemical reaction in a laboratory, for example) or intentionally modified is artificial.

As used herein, the term " parent " in the term " parent protein " or " parent domain " refers to an Ig binding protein that is subsequently modified to produce variants of the parent protein or domain. The parent protein or domain may be a naturally-occurring < RTI ID = 0.0 > staphylococcus < / RTI > (e. G., SEQ ID NOS: 3, 4, 10, 14, 21, 25, 47, 48, 49, Aureus protein A can be a domain, or a natural occurrence Staphylococcus aureus protein A, the mutant or manipulated version of the domain.

The term " variant " or " variant Ig binding domain " or " Ig binding domain variant " or " Ig binding protein variant " as used herein means an Ig binding protein or domain having at least one amino acid substitution, The parent protein or domain amino acid sequence and others. It also represents an artificial molecule different from the parent molecule by one or more modifications. The modification may be generated by genetic manipulation or chemical synthesis or chemical reaction performed by a human. For example, domain Z is a naturally occurring variant of protein A domain B. For example, SEQ ID NO: 30 is a variant of the maternal protein IB27.

The term " conjugate " as used herein relates to a molecule that comprises at least a molecule consisting essentially of, or consists essentially of, a first protein that is chemically attached to another substance, such as a second protein or a non-proteinaceous moiety.

The term " variant " or " amino acid modification " refers to the exchange, deletion, or insertion of an amino acid by another amino acid at a particular position in a parent polypeptide sequence. Given the known genetic code, and recombinant and synthetic DNA techniques, skilled scientists can readily produce DNAs encoding such amino acid variants.

The term " substitution " or " amino acid substitution " refers to the exchange of an amino acid by another amino acid at a particular position in a parent polypeptide sequence. For example, substitution S11A represents a variant Ig binding protein in which serine at position 11 has been replaced with alanine. In the previous example, 11A represents alanine at position 11. For purposes of this disclosure, multiple permutations are typically separated by a slash. For example, A1I / S11A / K35R represents a variant comprising a combination of substitutions A1I, S11A, and K35R.

The term " deletion " or " amino acid deletion " refers to the removal of an amino acid at a particular position in a parent polypeptide sequence.

The term " insert " or " amino acid insert " refers to the addition of an amino acid to a parent polypeptide sequence.

Throughout this specification, the amino acid residue numbering rules of FIG. 1 are used, and the position numbers are designated corresponding to, for example, SEQ ID NOS: 1-8.

The term " amino acid sequence identity " refers to a quantitative comparison of the identity (or difference) of amino acid sequences in two or more proteins. The terms " percent amino acid sequence identity " or " percent identity " or " percentage identity " with respect to the reference polypeptide sequence are based on the sequence of the reference polypeptide sequence, Is defined as the percentage of amino acid residues in the same sequence as the amino acid residue.

In order to determine sequence identity, the sequence of the query protein is aligned to the sequence of the reference protein. Alignment methods are well known in the art. For example, a freely available SIM local similarity program is preferably employed to determine the degree of identity of the amino acid sequence of any polypeptide relative to a reference amino acid sequence (Xiaoquin Huang and Webb Miller (1991), Advances in Applied Mathematics, vol. 12: 337-357) (see also http://www.expasy.org/tools/sim-prot.html). ClustalW is preferably used for multiple alignment assays (Thompson et al. (1994) Nucleic Acids Res., 22 (22): 4673-4680). Preferably, the SIM local similarity program or the default parameters of ClustalW are used when calculating percent sequence identity.

In the context of the present invention, unless expressly stated otherwise, the degree of sequence identity between the modified sequence and the sequence from which it is derived is generally calculated over the entire length of the unmodified sequence.

Each amino acid of the query sequence that differs from the reference amino acid sequence at a given position is counted as one difference. The sum of the differences then yields a percentage of non-identity relative to the length of the reference sequence. The quantitative identity percentage is calculated as a percentage of 100 minus non-identity.

As used herein, the phrase " percent identical " or " percent amino acid sequence identity " or " percent identity " refers to a polypeptide sequence that is determined using one of the following sequence comparison algorithms, , In some embodiments at least 89.5%, in some embodiments at least 91%, in some embodiments at least 93%, in some embodiments at least 94%, in some embodiments at least 95% 96% in some embodiments, at least 98% in some embodiments, and 100% in some embodiments nucleotide or amino acid residue identity. The percent identity may be determined for some regions of at least about 50 residues in some embodiments, for regions of at least about 51 residues in some embodiments, for regions of at least about 52 residues in some embodiments, For a region of at least about 53 residues, in some embodiments at least about 54 residues, in some embodiments at least about 55 residues, in some embodiments at least about 56 residues , In some embodiments about at least about 57 residues, and in some embodiments about at least about 58 residues. In some embodiments, percent identity is present for the entire length of the sequence.

The term " fused " means that the components are linked by peptide bonds, either directly or through a peptide linker.

The term " fusion protein " relates to a protein comprising at least a first protein that is genetically linked to at least a second protein. A fusion protein is originally formed through the binding of two or more genes that code for a particular protein. Thus, a fusion protein may comprise a multimer of the same or a different protein expressed as a single, linear polypeptide.

As used herein, the term " linker " refers in its broadest sense to a molecule that binds at least two other molecules in a covalent bond. In a typical embodiment of the present invention, a " linker " is understood as a moiety that links an Ig binding domain with at least one additional Ig binding domain, i.e., a moiety that links two protein domains to each other to form a multimer do. In a preferred embodiment, the " linker " is a peptide linker, i.e. the moiety linking two protein domains is a single single amino acid or a peptide comprising two or more amino acids.

The term " chromatography " refers to a separation technique that employs mobile and stationary phases to separate one type of molecules (e.g., immunoglobulins) from other molecules (e.g., contaminants) in a sample. The liquid mobile phase contains a mixture of molecules and transports them across or through a stationary phase (e.g., a solid matrix). Due to differential interactions between the different molecules in the mobile phase and the stationary phase, molecules can be separated in the mobile phase.

The term " affinity chromatography " means that the ligand coupled to a stationary phase interacts with a molecule (i.e., an immunoglobulin) in the mobile phase (i.e., a sample), that is, a specific manner in which the ligand has a specific binding affinity for the molecule to be purified ≪ / RTI > As will be understood in the context of the present invention, affinity chromatography involves adding a chromatographic ligand, e. G., A sample containing immunoglobulin to a stationary phase comprising an Ig binding protein of the invention.

The term " solid support " or " solid matrix " is used interchangeably for stationary phases.

The term " affinity matrix " or " affinity separation matrix " or " affinity chromatography matrix ", used interchangeably herein, refers to affinity ligands such as matrices to which the Ig binding proteins of the invention are attached, . A ligand (e.g., an Ig binding protein) can specifically bind to a molecule of interest (e.g., an immunoglobulin as defined above) to be purified or removed from the mixture.

The term " affinity purification " as used herein refers to a method of purifying an immunoglobulin as defined above from a liquid by binding an immunoglobulin as defined above to an Ig binding protein immobilized on a matrix. This removes all other components in the mixture other than the immunoglobulin. In an additional step, the bound immunoglobulin may be eluted in a purified form.

Invention Concrete example

The present invention will be further disclosed hereinafter. In the following paragraphs, various aspects of the present invention are defined in more detail. Unless expressly stated to the contrary, each aspect defined below may be combined with any other aspect or aspect. In particular, any feature pointed to as being preferred or advantageous may be combined with any other feature or feature pointed out as being advantageous or advantageous.

(Ig) binding protein comprising at least one Ig binding domain, wherein the at least one Ig binding domain comprises an amino acid substitution at position 1 to isoleucine, an alanine at position 11, 2, 3 or 4 substitutions selected from the group consisting of amino acid substitutions at amino acid positions 42 to 42, amino acid substitutions at amino acid positions 42 to 42, Or consist essentially of, or consist of, a variant of < RTI ID = 0.0 > In some embodiments, the at least one variant Ig binding domain further comprises 1, 2, 3, 4, 5, or 6 variants, each individual variant comprising a single amino acid substitution, a single amino acid deletion, Group.

The advantage of the variant Ig binding proteins is that they are stable for extended periods of time under alkaline conditions. This feature is important in a chromatographic approach with a cleaning procedure using an alkaline solution with a high concentration of NaOH, which can be used to remove contaminants on the matrix, for example, the matrix can be used several times. The variant Ig binding protein is more stable after alkaline treatment compared to the parent polypeptide. Substitution at position 1, 11, 35, and / or 42 in the parent protein, as defined above, provides improved alkaline stability without compromising immunoglobulin-binding properties compared to the parent protein.

Matrix SEQ ID NO: 1. In some embodiments, the immunoglobulin-binding protein has (i) SEQ ID NO: 1 or variants of the amino acid sequence of (ii) SEQ ID NO: at least the amino acid representing the 89.5% sequence identity to the amino acid sequence of the 1 And one or more Ig binding domains of a variant of the sequence. The Ig binding domain comprises an amino acid substitution at position 1 of isozyme at position 1 of SEQ ID NO: 1, an amino acid substitution at position 11 of alanine, glutamic acid, or isoleucine at position 11 of SEQ ID NO: 1, arginine or isoleucine at position 35 of SEQ ID NO: 2, 3, or 4 substitutions selected from the group consisting of amino acid substitutions of the amino acid sequence of SEQ ID NO: 1 with leucine at position 42 of SEQ ID NO: 1. The variant Ig binding protein may contain additional modifications, such as 1, 2, 3, 4, 5, or 6 substitutions or 1, 2, 3, 4, 5, or 6 deletions.

The Ig binding domain disclosed in SEQ ID NO: 1 is the mordant domain; The Ig binding domain having a substitution at least at positions 1, 11, 35, and / or 42 is a variant of SEQ ID NO: 1.

SEQ ID NO: 1 is an mature domain for variants of the present invention, preferably (i) an artificial Ig binding domain comprising SEQ ID NOS: 3, 4, 10, 14, 21, 25, 47, 48, 49, (ii) a consensus sequence comprising a naturally occurring protein A domain or variant comprising SEQ ID NOs: 5-8. The parent protein of SEQ ID NO: 1 is the following amino acid sequence:

X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ QQX ₁₁ AFYX ₁₅ X ₁₆ LX ₁₈ X ₁₉ PX ₂₁ LX ₂₃ X ₂₄ X ₂₅ QRX ₂₈ X ₂₉ FIQSLKDDPSX ₄₀ SX ₄₂ X ₄₃ X ₄₄ LX ₄₆ EAX ₄₉ KLX ₅₂ X ₅₃ X ₅₄ X ₅₅ APX ₅₈ wherein the amino acid (X ₁ ) is selected from P, N, A, V, or Q in position 1 and the amino acid (X ₂ ) is selected from the amino acid (X ₃₎ in position 3 is from the A, N, or S, preferably A or is selected from N, amino acid at position ₄ (X 4) X 4 is K or N, preferably a K The amino acid (X ₅ ) is selected from H or F at position 5 and the amino acid (X ₆ ) at position 6 is selected from D, N, A, or S, preferably D or N, The amino acid (X ₇ ) is selected from K or E, The amino acid at position ₈ (X 8) is D, A, or it is selected from E, the amino acid at position ₁₁ (X 11) is selected from S or N, the amino acid at position ₁₅ (X 15) is selected from E or Q , The amino acid (X ₁₆ ) at position 16 is selected from I or V, the amino acid (X ₁₈ ) at position 18 is selected from H or N and the amino acid (X ₁₉ ) X ₁₉ at position 19 is selected from L or M in the position 21 the amino acid (X ₂₁₎ is N, S, or D, preferably selected from N, amino acid at position ₂₃ (X 23) is selected from T or N, an amino acid (X ₂₄₎ at position 24 is E or A, the amino acid (X ₂₅ ) at position 25 is selected from D or E and the amino acid (X ₂₈ ) at position 28 is selected from S, N, or A, preferably N or S, 29, the amino acid (X ₂₉ ) is selected from A or G, the amino acid (X ₄₀₎ is V, Q, or T, preferably V or is selected from Q, where 42 amino acids (X ₄₂₎ is K, A, or is selected from T, the amino acid (X ₄₃₎ at the location 43 is (X ₄₄ ) is selected from I, V, or L, and the amino acid (X ₄₆ ) at position 46 is selected from G or A, wherein the amino acid is selected from E, N or S, preferably E or N; , located in the 49 amino acid (X ₄₉₎ is selected from K or Q, position 52 the amino acid (X ₅₂₎ is N, D, or S, preferably selected from N, an amino acid (X ₅₃₎ at the position 53 D or E, the amino acid (X ₅₄ ) at position 54 is selected from A or S, and the amino acid (X ₅₈ ) at position 58 is selected from P or K.

Matrix SEQ ID NO: 5 - 8. In one embodiment of the first aspect, the parent domain is SEQ ID NO: amino acid sequence, or SEQ ID NO: 5-8 of the amino acid representing at least 89.5% sequence identity to the amino acid sequence of 5 to 8 Or consist essentially of or consist of < RTI ID = 0.0 > SEQ ID < / RTI > The Ig binding domain comprises an amino acid substitution at position 1 to isoleucine, an amino acid substitution at position 11 to alanine, glutamic acid, or isoleucine, an amino acid substitution at position 35 to arginine or isoleucine, and an amino acid substitution at position 42 to leucine , Or at least one, two, three, or four amino acid substitutions selected from the group consisting of SEQ ID NOs. In some embodiments, the Ig binding domain further comprises 1, 2, 3, 4, 5, or 6 variants, wherein each individual variation is selected from the group consisting of a single amino acid substitution, a single amino acid deletion, do.

Matrix SEQ ID NO: 2. In another preferred embodiment of the first aspect, wherein the at least one Ig-binding domain is SEQ ID NO: contains a variant of the parent amino acid sequence of Figure 2, the variant of SEQ ID NO: isoleucine at position 1 of the 2 2, an amino acid substitution at position 11 of alanine, glutamic acid, or isoleucine at position 11 of SEQ ID NO: 2, an amino acid substitution of arginine or isoleucine at position 35 of SEQ ID NO: 2, At least 1, 2, 3 or 4 substitutions selected from the group consisting of amino acid substitutions to the amino acid sequence of SEQ ID NO. In some embodiments, the at least one Ig binding domain further comprises 1, 2, 3, 4, 5, or 6 variants, wherein each individual modification comprises a single amino acid substitution, a single amino acid deletion, Selected from the group consisting of.

SEQ ID NO: 2 is the consensus sequence for the preferred parent protein, including but not limited to the artificial Ig binding domains IB14, IB25, IB27, IB74, and IB47. Preferably, the invention relates to an Ig binding protein, wherein said at least one Ig binding domain comprises a variant of the amino acid sequence of the parental SEQ ID NO: 2 or a variant of the amino acid sequence having at least 89.5% identity to the parental SEQ ID NO: Or consist essentially of, or consist of. SEQ ID NO: 2 is a preferred embodiment of SEQ ID NO: 1: X ₁ AAX ₄ X ₅ DX ₇ X ₈ QQX ₁₁ AFYEILHLPNLTEX ₂₅ QRX ₂₈ AFIQSLKDDPSVSKEX ₄₄ LX ₄₆ EAX ₄₉ KLNDX ₅₄ QAPX _{58 In the} above, ₁₎ P, N, or is selected from A, the amino acid at position 5 (X ₅₎ is selected from H or F, and the amino acid (X in position ₇₇₎ is selected from K, or E, the amino acid at position 8 ( X ₈ is selected from D, A, or E; the amino acid (X ₁₁ ) at position 11 is selected from S or N, preferably S; and the amino acid (X ₂₅ ) at position 25 is selected from D or E , Amino acid (X ₂₈ ) at position 28 is selected from S or N, preferably N; amino acid (X ₄₄ ) at position 44 is selected from I or V; amino acid at position 46 (X ₄₆ ) It is selected from the amino acid at position ₄₉ (X 49) is selected from K or Q, at amino position 54 Acid (X ₅₄₎ is selected from A or S, and the amino acid at position ₅₈ (X 58) is selected from P or K.

Illustrative matrix proteins. In some embodiments, the Ig binding domain comprises a variant of a parental amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 4, 10, 14, 21, 25, 47-50, Or amino acid substitution of proline to isoleucine, alanine substitution of alanine, glutamic acid, or isoleucine at position 11, amino acid substitution of lysine to arginine or isoleucine at position 35, and amino acid substitution of lysine to leucine at position 42 At least 1, 2, 3, or 4 amino acid substitutions selected from the group consisting of.

IB14 as maternal protein . In a preferred embodiment of the first aspect, the parent protein is an amino acid sequence of SEQ ID NO: 3, or a protein having at least 89.5% identity to the parent SEQ ID NO: 3. Examples of the parent protein having at least 89.5% identity to SEQ ID NO: 3 include SEQ ID NO: 21 (1P / 28N), SEQ ID NO: 10 (1A / 28S), SEQ ID NO: 14 (1P / 28S) (5F / 7E / 8A / 25E), SEQ ID NO: 49 (44V / 49Q / 54S / 58K), SEQ ID NO: 5D / 7E / 8A / 28A), IB15 (2D / 3N / 5F / 7E / 8A / 28A), IB13 (1P / 21S / 28A / 40T / 43S) , And IB16 (2D / 3S / 5F / 7E / 8A / 28A).

IB27 as maternal protein . In another preferred embodiment of the first aspect, the parent protein is a protein having at least 89.5% identity to SEQ ID NO: 4, or to the parental SEQ ID NO: 4. An example for a parent protein having at least 89.5% identity is SEQ ID NO: 50 (5H / 7K / 8D), SEQ ID NO: 49 (5H / 7K / 8D / 25D); SEQ ID NO: 48 (44I / 49K / 54A / 58P), and SEQ ID NO: 47 (25D / 44I / 49K / 54A / 58P).

A more preferred matrix domain . In one embodiment of the first aspect, the parent protein is the amino acid sequence of SEQ ID NO: 25. In another embodiment of the first aspect, the parent protein is the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 49. In another embodiment of the first aspect, the parent protein is the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 47.

Preferred amino acid positions in alkaline stable proteins . In some embodiments, the Ig binding domain of a variant Ig binding protein comprises a substitution or multiple substitutions.

Replacement of the isoleucine (I) at position 1 may be the only substitution (e. G., SEQ ID NO: 9) or Ig-binding domain is part of the mutation, for example in the position 11 and / or 35 and / or 42 in the parent protein At least substitution. It is preferable that the amino acid at position 1 of the alkali-stable protein is not threonine (T). In position 1, the amino acid is preferably isoleucine (I) or alanine (A).

Substitution at position 11 for alanine (A), glutamic acid (E), or isoleucine (I) may be the only substitution (e. G., SEQ ID NO: 11-13) or the Ig binding domain is an additional mutation, May comprise at least substitutions at positions 1 and / or 35 and / or 42. [ It is preferred that the amino acid at position 11 is not asparagine (N) or lysine (K). The amino acid at position 11 is preferably alanine (A), isoleucine (I), glutamic acid (E), histidine (H) or proline (P), more preferably A, I or E and most preferably A. .

Substitution at position 35 for arginine (R) or isoleucine (I) may be the only substitution (e.g., SEQ ID NO: 15-16) or the Ig binding domain may be an additional mutation, preferably position 1 and / 11 and / or < RTI ID = 0.0 > 42. ≪ / RTI > Preferably, the amino acid at position 35 is not proline (P), asparagine (N), glycine (G), tryptophan (W), alanine (A), glutamine (Q), or methionine (M). The amino acid at position 35 is preferably R or I.

The substitution at position 42 for leucine (L) may be a unique substitution (e.g., SEQ ID NO: 17) or the Ig binding domain may be an additional mutation, preferably at position 1 and / or 11 and / Substitution. At position 42, it is preferred that the amino acid is not tyrosine (Y). The amino acid at position 42 is preferably L.

Preferred combinations of amino acids in the Ig binding domain . Surprisingly, certain combinations of amino acids at positions 1, 11, and 35, and optionally positions 1, 11, 35, 42 and optionally positions 1, 11, 28, 35, and 42, Likewise, the alkaline stability of the variant Ig binding domain is increased compared to the parental domain. In addition to substitution at positions 1, 11, 35, 42, the alkali stable Ig binding domain may comprise additional 1, 2, or 3 modifications such as substitution, deletion, or insertion. For example, at least one Ig binding domain comprises substitutions or substitutions compared to the parent sequence, and wherein said substitution or plurality of substitutions comprise: 1I; 11A; 35R; 42L; 11E; 11I; 35I; 11I / 11A; 1I / 35R; 11A / 35R; 1I / 42L; 11A / 42L; 11 / 11E; 11I / 11I; 11I / 35R; 11E / 35R; 11I / 42L; 11E / 42L; 1I / 35I; 11A / 35I; 11I / 35I; 11E / 35I; 35R / 42L; 35I / 42L; 1I / 11A / 35R; 1I / 11E / 35R; 1I / 11I / 35R; 1I / 11A / 42L; 1I / 11E / 42L; 1I / 11I / 42L; 1I / 11A / 35I; 1I / 11E / 35I; 1I / 11I / 35I; 1I / 35R / 42L; 1I / 35I / 42L; 11I / 35R / 42L; 11I / 35I / 42L; 11A / 35R / 42L; 11A / 35I / 42L; 11E / 35R / 42L; 11E / 35I / 42L; 1I / 11A / 35R / 42L; 1I / 11E / 35R / 42L; 1I / 11I / 35R / 42L; 1I / 11A / 35I / 42L; 1I / 11E / 35I / 42L; 1I / 11I / 35I / 42L; 1I / 11A / 28N / 35R / 42L; 1I / 11E / 28N / 35R / 42L; 1I / 11I / 28N / 35R / 42L; 1I / 11A / 28N / 35I, 42L; 1I / 11I / 28N / 35I / 42L; And 11 / 11E / 28N / 35I / 42L. 1I; 11A; 35R; 42L; 11I / 11A; 1I / 35R; 1I / 42L; 11A / 42L; 11A / 35R; 35R / 42L; 1I / 11A / 35R; 1I / 11A / 42L; 1I / 11A / 35R / 42L; And 11 / 11A / 28N / 35R / 42L. In some embodiments, 3 or 4 of the amino acid positions are selected from the group consisting of 1I, 11A, 35R, and 42L. In another embodiment, the Ig binding domain is selected from the group consisting of 1I / 11A / 35R; 1I / 11A / 35R / 42L; And a combination of substitutions selected from the group consisting of 1I / 11A / 28N / 35R / 42L.

In a preferred embodiment, the Ig binding protein of the invention comprises or consists essentially of one or more Ig binding domains, wherein the amino acid residue at position 1 is isoleucine and the amino acid residue at position 11 is alanine. Another preferred Ig binding protein of the invention comprises or consists essentially of one or more Ig binding domains wherein the amino acid residue at position 1 is isoleucine and the amino acid residue at position 11 is alanine, The amino acid residue is arginine. Another preferred Ig binding protein of the invention comprises or consists essentially of one or more Ig binding domains wherein the amino acid residue at position 1 is isoleucine and the amino acid residue at position 11 is alanine, The amino acid residue is arginine, and the amino acid residue at position 42 is leucine. Another preferred Ig binding protein of the invention comprises or consists essentially of one or more Ig binding domains wherein the amino acid residue at position 1 is isoleucine and the amino acid residue at position 11 is alanine, The amino acid residue is arginine, and the amino acid residue at position 42 is leucine, and the amino acid residue at position 28 is asparagine. Another preferred Ig binding protein of the invention comprises or consists essentially of one or more Ig binding domains wherein the amino acid residue at position 11 is alanine and the amino acid residue at position 35 is arginine and at position 42 The amino acid residue is leucine.

Sequence of an alkaline stable protein . The Ig binding protein of the present invention comprises at least one Ig binding domain comprising or consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: 52 . In some embodiments, the Ig binding domain comprises an amino acid sequence at least 89.5% identical to SEQ ID NO: 52. SEQ ID NO: 52 is a consensus sequence for the Ig-binding domain of a preferred artificial Ig binding protein, e. G., SEQ ID NOS: 18-20, 26, 29-30, 42-45, 56-61. SEQ ID NO: 52 is the following amino acid sequence (see Figure 1B):

IAAKX ₅ DX ₇ X ₈ QQAAFYEILHLPNLTEX ₂₅ QRX ₂₈ AFIQSLRDDPSVSX ₄₂ EX ₄₄ LX ₄₆ EAX ₄₉ KLNDX ₅₄ QAPX ₅₈ wherein the amino acid (X ₅ ) is selected from H or F at position 5 and the amino acid (X ₇ ) or it is selected from E, the amino acid at position ₈ (X 8) is selected from D, A, or E, the amino acid at position ₂₅ (X 25) is selected from D or E, position 28 is an amino acid (X ₂₈₎ from S or N, and the amino acid (X ₄₂ ) at position 42 is selected from L or K, preferably L; the amino acid (X ₄₄ ) at position 44 is selected from I or V; ₄₆₎ is selected from G or A, located in a 49 amino acid (X ₄₉₎ is selected from K or Q, the amino acid (X ₅₄ in position 54) is selected from A or S, and the amino acid at position ₅₈ (X 58 ) Is selected from P or K.

High binding stability as a result of a combination of 3 or 4 amino acids at position 1, 11, 35, 42 in the Ig binding protein . In some embodiments, the combination of at least three or four amino acids selected from isoleucine at position 1, alanine at position 11, arginine at position 35, and leucine at position 42, surprisingly, Particularly good alkaline stability of the binding protein. Position 28 is preferably asparagine. As disclosed in the following examples, all of the Ig binding proteins of the present invention were found to bind to Ig even after alkaline treatment. The Ig binding proteins of the present invention exhibit a high alkali stability, specifically improved alkali stability, for at least 6 hours at 0.5 M NaOH as compared to the corresponding parent protein.

Surprisingly and unexpectedly, there is provided a method for the production of at least three or four amino acids from amino acid position 1 to isoleucine, from amino acid position 11 to alanine, glutamic acid, or isoleucine, from amino acid position 35 to arginine or isoleucine and optionally from amino acid position 42 to leucine Binding Ig proteins can bind to Ig even after alkaline treatment for several hours. Most surprisingly and unexpectedly, the Ig binding proteins comprising a combination of the amino acids 11, 11A or 11E or 11I, 35R or 35I, and optionally a combination of 42L, preferably the amino acids 11, 11A, 35R and 42L, It can bind to Ig even after alkaline treatment. The alkali stability of the Ig binding proteins is determined by comparing the disappearance in Ig binding activity after incubation for 6 hours in 0.5 M NaOH. In some embodiments, this was compared to the loss of Ig-binding activity of the corresponding maternal protein. Loss of binding activity is determined by comparing binding activity before and after 0.5 M NaOH incubation for 6 hours.

The Ig binding activity of an Ig binding domain having at least 3 or 4 amino acids selected from 1I, 11A or 11E or 11I, 35R or 35I and 42L, as disclosed in the figure as comparison data, %. This is a surprising and beneficial characteristic compared to maternal proteins.

Preferred alkali stable Ig binding protein. In certain embodiments, the Ig binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 18-20, 26, 29-40, 42-45, and 56-61. In some embodiments, the domain comprises the amino acid sequence of a group consisting of SEQ ID NOS: 20, 26, 30, 42-45. The alkali stability domain may include additional modifications, such as insertion, deletion, or additional substitution. In some embodiments, the Ig binding domain has 1, 2, 3, 4, 5, or 6 additional substitutions. In other embodiments, the Ig binding domain has a deletion of one, two, three, or four amino acids within the first four amino acids of its N-terminus and / or deletion of one or two amino acids at the C-terminus. In some embodiments, the Ig binding domain has a deletion at the N-terminus, e.g., at positions 1, 2, and 4, or at positions 1, 2, and 3. In some embodiments, the Ig binding domain has a deletion at the C-terminus, e. G., Deletion at position 57 and / or 58. Some embodiments are directed to amino acids selected from the group consisting of SEQ ID NOs: 20, 26, 30, 42-45, including but not limited to SEQ ID NOs: 9-19, 29, 53-54, 56-61 RTI ID = 0.0 > 89.5% < / RTI > sequence identity. Some embodiments relate to an amino acid sequence having at least 89.5% sequence identity to the amino acid sequence for any of the above-identified sequence numbers, wherein the amino acid sequence having at least 89.5% sequence identity to any of the above sequence numbers is an amino acid sequence having the sequence At least three or four of the positions corresponding to the individual amino acid positions from which the amino acid sequences having positions 1, 11, 35 and 42 or at least 89.5% sequence identity in the sequence of SEQ ID NOs: 20, 26, 29, 30, Have the same amino acid. A sequence for a preferred alkali stable Ig binding protein is shown in FIG. At least three or four of the positions 1I, 11A, 35R, and 42L are preferably stored. It is also preferable that position 4 is not Q.

SEQ ID NO: 20 ( cs14 ) and variants. In certain embodiments, the alkali stable Ig binding domain comprises an amino acid sequence of SEQ ID NO: 20 or an amino acid sequence at least 91% identical thereto, for example, an amino acid sequence of SEQ ID NOs: 18-19, 26, 42-45, Or consist essentially of, or consist of. In a preferred embodiment, the variant of SEQ ID NO: 20 does not have Q at position 4. More preferably, the position 4 is K. In certain embodiments, the Ig binding domain comprises the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence that is at least 96% identical thereto. For example, Figures 3 and 5 showed the residual activity of Ig binding after extended sequential 0.5 M NaOH treatment.

Table 1 shows the amino acid differences of SEQ ID NO: 20 and at least 91% of the preferred variants thereof. Position 5 is H or F, position 7 is K or E, position 8 is D or A, position 25 is D or E, position 44 is I or V, position 46 is G or A, position 49 is K or Q, 54 is preferably A or S, and position 58 is preferably P or K. More preferably, the position 4 is K. The identity of the artificial alkaline stability to any wild-type protein A domain SEQ ID NO: 20 is equal to or less than 78%.

SEQ ID NO: 30 ( cs27 ) and variants. In certain embodiments, the Ig binding domain comprises or consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence at least 94% identical thereto, for example, the amino acid sequence of SEQ ID NO: 29 do. The identity of SEQ ID NO: 30 for any wild-type protein A domain is 76% or less. Figure 4 shows the remaining activity of SEQ ID NOS: 30 and 29 after 6 hours of continuous 0.5 M NaOH treatment.

SEQ ID NO: 26 ( cs25 ) and variants. In certain embodiments, the Ig binding domain comprises or consists essentially of or consists of the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence at least 98% identical thereto. The identity of SEQ ID NO: 26 to any wild-type protein A domain is 81% or less. Figure 5 shows the remaining Ig binding activity of SEQ ID NO: 26 after 6 hours of continuous 0.5 M NaOH treatment.

SEQ ID NO: 42 ( cs74 ) and variants. In certain embodiments, the alkali stable Ig binding domain comprises or consists essentially of or consists of the amino acid sequence of SEQ ID NO: 42 and an amino acid sequence at least 98% identical thereto, for example, the amino acid sequence of SEQ ID NO: . The identity of SEQ ID NO: 42 for any wild-type protein A domain is 78% or less. Figure 5 shows the remaining Ig binding of SEQ ID NO: 42-43 after 6 hours of continuous 0.5 M NaOH treatment.

SEQ ID NO: 44 ( cs47 ) and variants thereof. In certain embodiments, the alkali stable Ig binding domain comprises or consists essentially of the amino acid sequence of SEQ ID NO: 44 and an amino acid sequence at least 98% identical thereto, for example, the amino acid sequence of SEQ ID NO: 45. The identity of SEQ ID NO: 44 for any wild-type protein A domain is 78% or less. Figure 5 shows the remaining activity of Ig binding after 6 hours of continuous 0.5 M NaOH treatment of SEQ ID NO: 44-45 with at least 98% identity.

Affinity for immunoglobulins. All Ig-binding proteins of the invention bind immunoglobulin with a dissociation constant, K _D , preferably no more than 1 μM, or no more than 100 nM, more preferably no more than 10 nM. Methods for determining the binding affinity of an Ig binding protein or domain, i. E. For determining the dissociation constant, K _D , are known to those of ordinary skill in the art and can be found, for example, : Surface plasmon resonance (SPR) -based technology, bio-layer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), flow cytometry flow cytometry, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA), and enhanced chemiluminescence (ECL). Some methods are further disclosed in the examples. Typically, the dissociation constant K _D is determined at 20 占 폚, 25 占 폚, or 30 占 폚. Unless otherwise specifically indicated, the K _D values quoted herein are determined by surface plasmon resonance at 22 ° C +/- 3 ° C. In one embodiment of the first aspect, the Ig binding protein has a dissociation constant K _D for human IgG ₁ ranging from 0.1 nM to 100 nM, preferably from 0.1 nM to 10 nM.

Multimers. In one embodiment of the invention, the Ig binding protein comprises 1, 2, 3, 4, 5, 6, 7, or 8, preferably 2, 3, 4, 5, Domain, i.e., the Ig binding protein can be, for example, a monomer, a dimer, a trimer, a tetramer, a trimer, or a mass. The oligomer may comprise 2, 3, 4, or more binding domains.

The oligomers of the present invention are generally fusion proteins that are artificially produced by recombinant DNA techniques well known to those skilled in the art. The Ig binding proteins of the present invention can be prepared using any of the many existing and well known techniques such as plain organic synthetic strategies, solid phase-assisted synthesis techniques, ≪ / RTI >

In some preferred embodiments, the oligomers are homo-multimers, e.g., the amino acid sequences of all the alkali stable Ig binding domains in the Ig binding protein are the same.

The alkali-stable multimer may comprise two or more Ig binding domains and the Ig binding domain is preferably selected from the group consisting of SEQ ID NOS: 18-20, 26, 29-38, 42-45, 56-61 Or a sequence having at least 89.5% sequence identity to any of the foregoing sequence numbers. In some embodiments, the domain is a derivative of SEQ ID NOS: 18-20, 26, 29-38, 42-45, 56-61 and further wherein each derivative is selected from the group consisting of SEQ ID NOs: 18-20, 26, 29-38, Deletion of one, two or three amino acids in the first four amino acids of its N-terminus and / or deletion of one or two amino acids at the C-terminus thereof for one of the amino acids 42-45, 56-61 For example, SEQ ID NOS: 23, 24 and 27).

For example, SEQ ID NO: 14 and SEQ ID NO: 20 were used to generate the homo-multimer fusion constructs (dimers, tetramers, dimers, and tumors) disclosed herein in Example 1. Also, dimer, tetramer, pentamer, and hemimers of SEQ ID NO: 30 were produced. For example, see SEQ ID NO: 23, 24, 27,

In some embodiments of the first aspect, the multimer is heterodimeric, e.g., at least one of the alkaline stable Ig binding domains has an amino acid sequence that differs from other Ig binding domains in the immunoglobulin-binding protein.

Linker. In some embodiments of the first aspect, the one or more Ig binding domains are directly linked to each other. In another embodiment, the one or more Ig binding domains are linked to each other by one or more linkers. In the above conventional embodiments, it is preferable that the peptide linker is a peptide linker. This means that the peptide linker is an amino acid sequence linking the first Ig binding domain with the second Ig binding domain. The peptide linker is linked to a first Ig binding domain and a second Ig binding domain by a peptide bond between the C-terminus and the N-terminus of the domains to produce a single, linear polypeptide chain. The length and composition of the linker can vary between at least one and up to about 30 amino acids. More specifically, the peptide linker has a length of 1 to 30 amino acids; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30 amino acids. It is preferable that the amino acid sequence of the peptide linker is stable to the corrosion condition and the protease. The linker should not render the conformation of the domain unstable among the Ig binding proteins. Linkers containing small amino acids such as glycine and serine are well known. The linker may be glycine-rich (e.g., greater than 50% of the residues in the linker may be a glycine residue). Linkers containing additional amino acids are also preferred. Other embodiments of the invention include a linker consisting of alanine, proline, and serine. Other linkers for fusion of proteins are known in the art and may also be used.

Conjugation to a solid support . In some embodiments of the invention, the Ig binding protein is conjugated to a solid support. In some embodiments of the invention, the Ig binding domain comprises an attachment site for site-specific covalent coupling of the Ig binding protein to a solid support. In some embodiments of the invention, the Ig binding protein also includes additional amino acid residues at the N- and / or C-terminal end, such as a leader sequence at the N-terminal end and / or N- or C - contain a coupling sequence with or without a tag at the terminal end (see, e.g., SEQ ID NOS: 39 and 40). In some embodiments, the alkali stable Ig binding protein comprises an attachment site for covalent attachment to a solid phase (matrix). Preferably, the attachment site is specific to provide site-specific attachment on the solid. Certain attachment sites include neutral amino acids such as cysteine or lysine, which may be in the form of a solid phase reactor or solid phase and protein, for example, selected from N-hydroxysuccinimide, iodoacetamide, maleimide, epoxy, Lt; RTI ID = 0.0 > linker. ≪ / RTI > The attachment site may be directly at the C- or N-terminal end of the Ig binding protein or may be a linker between the N- or C-terminus and the coupling site, preferably a peptide linker. In some embodiments of the invention, the Ig binding protein can comprise a short N- or C-terminal peptide sequence of 3 to 20 amino acids, preferably 4 to 10 amino acids, with terminal cysteine. The amino acid for the C-terminal attachment site can preferably be selected with proline, alanine, and serine, e.g., ASPAPSAPSAC (SEQ ID NO: 41), with a single cysteine at the C-terminal end for coupling. In other embodiments, the amino acid for the C-terminal attachment site can be selected, preferably with glycine and serine, for example, GGGSC, with a single cysteine at the C-terminal end for coupling.

The advantage of having C-terminal cysteine is that the coupling of the Ig binding protein can be achieved by inducing thioether bridge coupling through the reaction of the cysteine thiol with the electrophilic group on the support. This provides excellent mobility of the coupled protein providing increased binding capacity.

In alternative embodiments, the coupling of the Ig binding protein to a solid support can be accomplished via cysteine at positions 43, 46, or 47 of the alkali stable Ig binding domain. When cysteine is located at position 43, 46, or 47, the amino acid at position 50 or position 58 is not cysteine (see, e.g., SEQ ID NO: 53 or 54). Preferably the amino acid at position 50 is lysine and the amino acid at position 58 is selected from proline or lysine.

Affinity separation matrix. In another aspect, the invention is directed to an affinity separation matrix comprising an Ig binding protein of the first aspect.

In a preferred embodiment of the second aspect, the affinity separation matrix is a solid support. The affinity separation matrix comprises at least one Ig binding protein of the present invention.

The matrix comprising an alkali stable Ig binding protein of the invention comprises an immunoglobulin as defined above, i.e. an Ig, an Ig variant comprising an Fc region, a fusion protein comprising an Fc region in Ig, and an Fc region in Ig Is useful for separation of the conjugate, e. G. Chromatographic separation. The affinity matrix will be useful for the separation of immunoglobulins and will retain Ig binding properties even after severe alkaline conditions applied during the cleansing process. This cleaning of the matrix is necessary for long-term repeated use of the matrix.

Solid support matrices for affinity chromatography are known in the art and include, but are not limited to, stabilized derivatives of agarose and agarose (e.g., Sepharose 6B, Praesto ^TM Pure; CaptivA ^® , rPROTEIN a Sepharose Fast Flow, Mabselect ^®, etc.), cellulose or cellulose derivatives, controlled pore glass (for example, ProSep ^® vA resin), the monolith (monolith) (e.g. CIM ^® monolithic), silica, zirconium oxide (for example CM Zirconia or CPG ^®), titanium oxide, or synthetic polymers (for example polystyrene, for example Poros 50A or Poros MabCapture ^® A resin, polyvinyl ether, polyvinyl alcohol, poly hydroxyalkyl acrylates, poly hydroxyalkyl methacrylates, poly acrylamides, Polymethacrylamide, etc.) and hydrogels of various compositions. In certain embodiments, the support comprises a polyhydroxy polymer, such as a polysaccharide. Examples of suitable polysaccharides for the support include, but are not limited to, agar, agarose, dextran, starch, cellulose, pullulan and the like and stabilized variants thereof.

The format of the solid support matrix may be any suitable widely known kind. Such solid support matrices for the coupling of the Ig binding proteins of the present invention can be used for example in columns, capillaries, particles, membranes, filters, monoliths, fibers, pads, gels, slides, plates, cassettes, And may include any of the other formats known to those skilled in the art.

In one embodiment, the matrix is composed of substantially spherical particles, also known as beads, e.g., Sepharose or agarose beads. Suitable particle sizes may range from 5-500 [mu] m, such as 10-100 [mu] m, such as 20-80 [mu] m in diameter. The particulate matrix may be used in a suspended form including a packed bed or an extended bed.

In an alternative embodiment, the solid support matrix is a membrane, e. G., A hydrogel membrane. In some embodiments, the affinity purification comprises a membrane as a matrix in which the alkali stable Ig binding protein of the first aspect is covalently bonded. The solid support may also be in the form of a membrane in the cartridge.

In some embodiments, the affinity purification comprises a chromatography column comprising a solid support matrix wherein the alkali stable Ig binding protein of the first aspect is covalently bonded.

The alkali stable Ig binding proteins of the present invention can be attached to suitable solid support matrices through conventional coupling techniques using, for example, amino-, sulfhydryl-, and / or carboxy-groups present in the Ig binding proteins of the invention have. The coupling can be carried out via the nitrogen, oxygen, or sulfur atom of the Ig binding protein. Preferably, the amino acid contained in the N- or C-terminal peptide linker comprises said nitrogen, oxygen or sulfur atom.

The Ig binding protein is coupled to the support matrix either directly or via a spacer element indirectly to improve the availability of the Ig binding protein and to facilitate the chemical coupling of the Ig binding protein of the invention to the support. And an Ig binding protein of the present invention.

Methods for immobilizing protein ligands to solid supports are well known in the art and are readily accomplished by those skilled in the art using standard techniques and equipment.

Depending on the Ig binding protein and the specific conditions, the coupling may be, for example, multipoint coupling through several lysines, or single point coupling via, for example, cysteine.

Use of an alkali stable Ig binding protein . In a third aspect, the invention relates to the use of an alkali stable Ig binding protein of the first aspect or an affinity matrix of the second aspect for affinity purification of immunoglobulins or variants thereof, i.e. the Ig binding proteins of the invention are affinity Is also used for chromatography. In some embodiments, an Ig binding protein of the invention is immobilized on a solid support as disclosed in the second aspect of the invention.

A method for purifying the affinity of an immunoglobulin . In a fourth aspect, the present invention relates to a method of purifying the affinity of an immunoglobulin, comprising the steps of: (a) providing a liquid containing an immunoglobulin; (b) providing an affinity separation matrix comprising an immobilized alkali stable Ig binding protein of a first aspect coupled to an affinity separation matrix; (c) contacting the liquid with the affinity separation matrix, wherein the immunoglobulin binds to the immobilized Ig binding protein; And (d) eluting the immunoglobulin from the matrix, thereby obtaining an eluate containing the immunoglobulin. In some embodiments, the affinity purification method further comprises, between steps (c) and (d), contacting the affinity separation matrix under conditions sufficient to remove some or all of the non-specifically bound molecules from the affinity separation matrix And may further include one or more cleaning steps. Non-specifically binding means any binding that does not involve the interaction between the binding domain of at least one of the subject matter disclosed herein and the immunoglobulin.

Affinity separation matrices suitable for the disclosed uses and methods are those known to those skilled in the art according to the embodiments described above.

In some embodiments of the fourth aspect, the elution of the immunoglobulin from the matrix in step (d) is performed through a change in pH and / or a change in salt concentration. Any suitable solution used for elution from the protein A medium, for example a solution of pH 5 or less, or a solution of pH 11 or higher, may be used.

In some embodiments, a further step (f) for effectively clearing the affinity matrix is preferably added, for example using an alkaline solution of pH 13-14. In certain embodiments, the rinse solution comprises 0.1-1.0 M NaOH or KOH, preferably 0.25-0.5 M NaOH or KOH. Due to the high alkaline stability of the Ig binding proteins of the present invention, this strong alkaline solution can be used for cleaning purposes.

In some embodiments, the affinity matrix is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 20, at least 30, at least 40, at least 60, 70, at least 80, at least 90, or at least 100 times, optionally (a) to (f) being at least 10, at least 20, at least 30, at least 40, at least 50, At least 60, at least 70, at least 80, at least 90, or at least 100 times.

In general, suitable conditions for carrying out the affinity purification method are well known to those skilled in the art, particularly those skilled in the protein A chromatography.

Nucleic acid molecule. In a fifth aspect, the invention relates to a nucleic acid molecule, preferably an isolated nucleic acid molecule, encoding an alkaline stable Ig binding protein of any of the embodiments described above. In one embodiment, the invention relates to a vector comprising a nucleic acid molecule. A vector refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) that can be used to deliver protein coding information to a host cell. In one embodiment, the vector is an expression vector.

In a sixth aspect, the present invention relates to an expression system comprising a nucleic acid or a vector as described above, for example a prokaryotic host cell, e . Coli (E. coli), or eukaryotic host, such as yeast Saccharomyces Mrs. Serenity busy (Saccharomyces cerevisiae ) or Pichia Paz pastoris (Pichia pastoris), or mammalian cells for example, relates to a CHO cell.

A method for the production of an alkali stable Ig binding protein . In a seventh aspect, the present invention relates to a method for the production of an alkali stable Ig binding protein of the invention, said method comprising the steps of: (a) contacting the expression of the binding protein to obtain an alkaline stable Ig binding protein Culturing the host cell of the sixth aspect under suitable conditions; And (b) optionally isolating the alkali stable Ig binding protein.

Suitable conditions for culturing prokaryotic or eukaryotic hosts are well known to those skilled in the art.

The Ig-binding molecules of the present invention may be prepared by any of a variety of conventional and well known techniques, such as basic organic synthesis strategies, solid phase-assisted synthesis techniques, or commercially available automated synthesizing devices. On the other hand, they can also be produced by conventional recombinant techniques alone or in combination with existing synthetic techniques.

One embodiment of the present invention is directed to a method of preparing an alkaline-stable Ig binding protein according to the present invention as described above, said method comprising the steps of: (a) reacting a nucleic acid encoding an Ig binding protein as defined above Lt; / RTI > (b) introducing the nucleic acid into an expression vector; (c) introducing the expression vector into a host cell; (d) culturing the host cell; (e) subjecting the host cell to a culture condition in which an Ig binding protein is expressed, thereby producing (e) an Ig binding protein as described above; Optionally (f) isolating the protein produced in step (e); And (g) optionally conjugating the protein to the solid matrix described above.

In a further embodiment of the present invention, the production of the alkali stable Ig binding protein is carried out by bit-by-cell transcription / translation without a cell.

Example

The following examples are provided for further illustration of the present invention. However, the present invention is not limited thereto, and the following examples also illustrate the feasibility of the present invention based only on the above description.

Example  One. On shuffling  Generation of maternal protein by

The parent protein (e. G., SEQ ID NOS: 3, 4, 10, 14, 21, 22, 25, 47-50) is first introduced into the naturally occurring protein A domain and the protein A domain variant (e. G., Z domain or any naturally occurring domain Such as the Z / 2 domain, having at least 89.5% identity to the amino acid sequence of SEQ ID NO: 2). More specifically, the shuffling process as understood herein is an assembly process that derives an artificial amino acid sequence, starting from a set of non-identical known amino acid sequences. The shuffling process comprises the steps of: a) providing a sequence of five naturally occurring protein A domains E, B, D, A, and C, and a protein A variant domain Z or Z / 2; b) aligning the sequences; c) identifying the subsequence to be recombined with in silico statistical fragmentation, and then d) assembling the new, artificial sequence of the various fragments to produce a mosaic product, i.e. a novel amino acid sequence. The fragment produced in step c) has an arbitrary length, for example, when the fragmented parent sequence has a length of n, the fragment has a length of 1 to n-1.

The relative position of the amino acids in the mosaic product was maintained for the starting amino acid sequence. The positions Q9, Q10, A12, F13, Y14, L17, P20, L22, Q26, R27, F30, I31, Q32, S33, L34, K35, D36, D37, P38, S39, S41, L45, E47, , At least 90% of L51, Q55, A56, P57 is between the artificial amino acid sequence of IB14, IB25, IB74h1, IB74h2, IB47h3, IB47h4, and / or IB27 and the naturally occurring protein A domain or protein A domain variant . The entire amino acid sequence of the Ig binding proteins IB14, IB25, IB74h1, IB74h2, IB47h3, IB48h4, and IB27 is an artificial sequence in that it is equal to or less than 85% of the entire amino acid sequence of the naturally occurring protein A domain or domain Z. After the initial artificial Ig binding protein is generated, the protein is further modified by site-specific randomization of the amino acid sequence to further modify the binding properties. These additional modifications are introduced by site-saturation mutagenesis of individual amino acid residues.

Ig binding protein IB14, IB25, IB47, IB74 / or IB27, as well as SEQ ID NO: 5-8 of the gene is synthesized using standard methods known to those skilled in the art and are. Collier was cloned into the expression vector. DNA sequencing was used to confirm the correct sequence of inserted fragments.

Two, three, four, five, or six identical Ig binding domains (e. G., SEQ ID NOS: 14, 20, 30) were genetically fused through an amino acid linker to produce a multimeric Ig binding protein.

(ASPAPSAPSAC; SEQ ID NO: 41) and optionally a strep-tag (WSHPQFEK; SEQ ID NO: 46) with C-terminal Cys were added to the C-terminus of the Ig binding protein for specific membrane attachment and purification (See, for example, SEQ ID NO: 39-40). In another embodiment, position 43, 46, or 47 is replaced by cysteine (see, e. G., SEQ ID NOs: 53-54) for specific membrane attachment and purification.

Example  2. Mutant  To generate Mutation

For site-directed mutagenesis, the Q5 ^® site-specific mutagenization kit (NEB; Cat. No. E0554S) was used according to the manufacturer's instructions. PCRs were performed using oligonucleotides each coding for each specific substitution and a plasmid comprising SEQ ID NO: 14 as template. The products were ligated and transformed by electroporation into E. coli XL2-blue cells (Stratagene). Single colonies were isolated and DNA sequencing was used for inserts containing clones to confirm the correct sequence. The results are shown in Fig.

A combination of several point mutations was generated using the GeneArt ^TM Strings ^TM synthesis (Thermo Fisher Scientific). The Strings DNA fragment corresponded to the purified PCR product and was cloned into a derivative of the pET28a vector. Ligation product . Coli XL2-blue cells were electroporated. A single colony was screened by PCR to identify the construct containing the correct size insert. DNA sequencing was used to confirm the correct sequence. Mutants with point mutations are disclosed, for example, in SEQ ID NOS: 9-13, 15-17, and 21.

Example  3. Expression of Ig binding proteins

BL21 (DE3) competent cells were transformed with an expression plasmid encoding an Ig binding protein. Cells were developed on selective agar plates (kanamycin) and incubated overnight at 37 < 0 > C. Pre-cultures were inoculated from a single colony in 100 ml 2xYT medium and cultured in a baffled 1 L Erlenmeyer flask supplemented with 150 [mu] g / ml kanamycin containing no lactose and defoamer at 160 rpm at 160 rpm in a conventional orbital shaking apparatus Lt; / RTI > for 16 hours. OD ₆₀₀ readings should be in the 6-12 range. The primary cultures were suspended in 400 ml superrich medium (modified H15 medium 2% glucose, 5% yeast extract, 0.89% glycerol, 0.76% lactose, 250 mM MOPS, 202 mM TRIS, pH 7.4, defoamer SE15) inoculated from a previous overnight culture adjusted to an on-OD ₆₀₀ of 0.5. The culture was transferred to a resonance acoustic mixer (RAMbio) and incubated at 37 ° C with 20 x g. Oxy-Pump stopper facilitated ventilation. After glucose metabolism, lactose was introduced into the cells to induce recombinant protein expression. OD ₆₀₀ was measured at a predetermined time point, a sample adjusted to 5 OD ₆₀₀ was drawn out, pelletized and frozen at -20 ° C. The cells were grown overnight for approximately 24 hours to reach a final OD ₆₀₀ of about 45-60. To collect the biomass, the cells were centrifuged at 16000 xg for 10 minutes at 20 < 0 > C. The pellet was weighed (wet weight) and the pH was measured in the supernatant. Cells were stored at -20 ° C until treatment.

Example  4: Expression and Solubility of Ig Binding Proteins SDS -PAGE analysis

Samples taken during fermentation were resuspended in 300 μl extraction buffer (0.2 mg / ml lysozyme, 0.5 x BugBuster, 7.5 mM MgSO ₄ , PBS supplemented with 40 μl benzene) and incubated for 15 min at 700 rpm in a thermomixer Which was solubilized by shaking. Soluble proteins were separated from insoluble proteins by centrifugation (16000 xg, 2 min, rt). The supernatant was withdrawn (soluble fraction) and the pellet (insoluble fraction) was resuspended in an equal volume of urea buffer (8 M urea, 0.2 M Tris, 2 mM EDTA, pH 8.5). 50 [mu] l from both the soluble and insoluble fractions were taken and 5 [mu] l of 0.5 M DTT as well as 12 [mu] l of 5x sample buffer were added. The sample was boiled at 95 ° C for 5 minutes. Finally, 8 μl of these samples were applied to NuPage Novex 4-12% Bis-Tris SDS gels, performed according to the manufacturer's recommendations and stained with Coomassie. High expression levels of all Ig binding proteins were confirmed under optimized conditions within the selected time period (data not shown). All expressed Ig binding proteins were more than 95% soluble by SDS-PAGE.

Example  5: Purification of Ig binding protein

Ig binding protein with the C-terminal StrepTagII (WSHPQFEK; SEQ ID NO: 46) . RTI ID = 0.0 > soluble fraction. ≪ / RTI > Cells were lysed in two freeze / thaw cycles and purification steps were performed on Strep-Tactin ( ^R ) resin (IBA, Goettingen, Germany) according to the manufacturer's instructions. To avoid disulfide formation, the buffer was supplemented with 1 mM DTT.

Alternatively, the Ig binding protein may be modified with the C-terminal StrepTagII (SEQ ID NO: 46) . RTI ID = 0.0 > soluble fraction. ≪ / RTI > Cells were resuspended in cell destruction buffer and lysed by constant cell disruption system (Unit F8B, Holly Farm Business Park) at 1 kbar for two cycles. The purification step was performed by Strep-Tactin-resin (IBA, Goettingen, Germany) and further gel filtration (Superdex 75 16/60; GE Healthcare) using the AKTAxpress system (Ge Healthcare) according to the manufacturer's instructions. To avoid disulfide formation, the buffer for Strep-Tactin-purification was supplemented with 1 mM DTT and citrate-buffer (20 mM citrate, 150 mM NaCl, pH 6.0) was added to the run buffer for gel filtration running buffer).

Example  6. Ig binding High protein  Friendly road To IgG  Combination (determined by ELISA)

The affinities of Ig binding proteins for IgG ₁ or IgG ₂ or IgG ₄ were determined using enzyme linked immunosorbant assay (ELISA). IgG ₁ or IgG ₂ or IgG ₄ containing antibodies (eg, cetuximab for IgG ₁ , panituum mit for IgG ₂ , or natalizumab for IgG ₄ ) on a 96 well Nunc MaxiSorb ELISA plate (2 μg / ml) . After incubation at 4 ° C for 16 hours, the wells were washed three times with PBST (PBS + 0.1% Tween 20) and the wells blocked with 3% BSA in PBS (2 hours at room temperature). Negative controls were Wells blocked only with BSA. After blocking, the wells were washed 3 times with PBST and incubated with Ig binding protein (in PBST) for 1 hour at room temperature. After incubation, the wells were washed three times with PBST and incubated with Strep-Tactin-HRP (1: 10000) (IBA, Goettingen, Germany) for 1 hour at room temperature. The wells were then washed three times with PBST and three times with PBS. The activity of horseradish peroxidase was visualized by the addition of TMB-Plus substrate. After 30 minutes, the reaction was stopped by the addition of 0.2 MH ₂ SO ₄ and the absorbance at 450 nm was measured. As determined by ELISA, the K _D for human IgG ₁ is 4.9 nM for SEQ ID NO: 14; 3.4 nM for domain Z; 3.1 nM for domain B; And 2.8 nM for Domain C, respectively.

Example  7. Ig binding High protein  Friendly road To IgG  Coupling box Plasmon  Determined by resonance experiments)

The CM5 sensor chip (GE Healthcare) was equilibrated to the SPR running buffer. The surface-exposed carboxyl groups were activated by passing through a mixture of EDC and NHS to yield a reactive ester group. 700-1500 RU on-immobilized on the ligand (ligand on-) a flow cell (flow cell), and the off-was immobilized ligand (off- ligand) on a different flow cell. After ligand immobilization, the non-covalently bound Ig binding protein was removed by the injection of ethanolamine. Upon ligand binding, a protein analyte was accumulated on the surface to increase the refractive index. These refractive index changes were measured in real time and plotted with time-response or resonance unit (RU). The analytes were serially diluted at the appropriate flow rate ([mu] l / min) and applied to the chip. After each run, the chip surface was regenerated as a regeneration buffer and equilibrated to the running buffer. A control sample was applied to the matrix. Regeneration and re-equilibration were performed as previously mentioned. Binding studies were performed using Biacore ^® 3000 (GE Healthcare) at 25 ° C; Data evaluation was performed using BIAevaluation 3.0 software, provided by the manufacturer, using a Langmuir 1: 1 model (RI = 0). The estimated dissociation constant (K _D ) was normalized to the off-target and the various artifacts for human IgG ₁ -Fc, cetuximab (IgG ₁ ), natalizumab (IgG ₄ ), or panitumomab (IgG ₂ ) The K _D values of the alkali stable Ig binding proteins are shown in Table 2 below.

K _D value of Ig binding protein to Ig SEQ ID NO: Ig binding protein IgG1 (nM) IgG4 (nM) IgG2 (nM) 20 cs14 2.9 2.51 7.42 30 cs27 3.64 2.54 21.6 26 cs25 4.24 3.27 11.6 45 cs47H4 4.1 3.11 25.2 44 cs47H3 4.78 4.05 20.3 43 CS74H2 3.48 2.72 17.2 42 cs74h1 1.64 1.2 12.8

Example  8. In an epoxy-activated matrix Coupled  Alkali stability of Ig binding proteins

The purified Ig-binding protein was added to an epoxy-activated matrix (Sepharose 6B, GE; Cat. No. 17-0480-01) according to the manufacturer's instructions (coupling conditions: pH 9.0 overnight, blocked with ethanolamine for 5 hours) Lt; / RTI > Cetuximab was used as an IgG sample (5 mg; 1 mg / ml matrix). Cetuximab was applied as a saturating amount to a matrix containing immobilized Ig binding protein. The matrix was washed with 100 mM glycine buffer, pH 2.5 to elute cetuximab bound to the immobilized IgG-binding protein. The concentration of IgG eluted to determine the binding activity of the Ig binding protein was measured by BLI (quantified as Protein A Octet-sensor and Cetuximab standard). The column was incubated with 0.5 M NaOH at room temperature (22 [deg.] C +/- 3 [deg.] C) for 6 hours. The Ig binding activity of the immobilized protein was analyzed before and after incubation with 0.5 M NaOH for 6 hours. The Ig binding activity of the immobilized protein before NaOH treatment was defined as 100%.

Figure 2 shows an analysis of the alkali stability of point mutants of IB14 (SEQ ID NO: 14). The remaining activity (%) of Ig binding after 6 hours of continuous 0.5 M NaOH treatment of point mutations at positions 1, 11, 35, and 42 of IB14 was compared to that of maternal IB14. Substitution PI, S11E, S11I, S11A, K35I, K35R, or K42L improved Ig binding activity by at least about 25%.

Figure 3 shows the activity of a variant protein having a combination of 3, 4, or 5 substitutions at positions 1, 11, 28, 35, and / or 42 for example compared to the activity of the maternal protein IB14 (SEQ ID NO: 14) And showed higher. (SEQ ID NO: 18) is about at least 50%, cs14-2 (SEQ ID NO: 19) is about at least 70%, and cs14 -3 (SEQ ID NO: 20) showed at least 80% higher Ig binding activity.

Figure 4 shows that the activity of variant Ig binding proteins with combinations of 3, 4, or 5 substitutions at positions 1, 11, 35, and / or 42 is higher compared to the activity of the maternal protein IB27. Approximately at least 30% of cs27-1 (SEQ ID NO: 29) and cs27-2 (SEQ ID NO: 30) are at least about 40% higher than the parental protein after incubation in 0.5 M NaOH for 6 hours Activity.

Figure 5 shows the activity of variant Ig binding proteins with combinations of 1I, 11A, 35R, and 42L compared to the activity of a maternal protein denoted here IB14 (the other parent protein being comparable to IB14, data not shown) Showed higher. (SEQ ID NO: 42), cs74h2 (SEQ ID NO: 43), cs47h3 (SEQ ID NO: 44), and cs47h4 (SEQ ID NO: 45) as compared to IB14 after incubation in 0.5 M NaOH for 6 hours. , And cs25 (SEQ ID NO: 26) showed significantly higher Ig binding activity.

                         SEQUENCE LISTING <110> Navigo Proteins GmbH   <120> Alkaline stable Immunoglobulin-binding proteins <130> SP32 WO <160> 55 <170> PatentIn version 3.5 <210> 1 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> generic sequence 1 <220> <221> variant <222> (1) <223> may be replaced by V, N, Q, or P <220> <221> variant <222> (2) (2) <223> may be replaced by D or Q <220> <221> variant &Lt; 222 > (3) <223> may be replaced by N or S <220> <221> variant <222> (4) (4) <223> may be replaced by N <220> <221> variant &Lt; 222 > (5) <223> may be replaced by F <220> <221> variant <222> (6) <223> may be replaced by N, A, or S <220> <221> variant <222> (7) (7) <223> may be replaced by E <220> <221> variant &Lt; 222 > (8) <223> may be replaced by E ore <220> <221> variant &Lt; 222 > (11) <223> may be replaced by N <220> <221> variant &Lt; 222 > (15) <223> may be replaced by Q <220> <221> variant &Lt; 222 > (16) <223> may be replaced by V <220> <221> variant <222> (18). (18) <223> may be replaced by N <220> <221> variant <222> (19). (19) <223> may be replaced by M <220> <221> variant &Lt; 222 > (21) <223> may be replaced by S or D <220> <221> variant &Lt; 222 > (23) <223> may be replaced by N <220> <221> variant &Lt; 222 > (24) <223> may be replaced by A <220> <221> variant &Lt; 222 > (25) <223> may be replaced by E <220> <221> variant &Lt; 222 > (28) <223> may be replaced by S ore <220> <221> variant <222> (29). (29) <223> may be replaced by G <220> <221> variant &Lt; 222 > (40) <223> may be replaced by Q or T <220> <221> variant &Lt; 222 > (42) <223> may be replaced by A or T <220> <221> variant &Lt; 222 > (43) <223> may be replaced by N or S <220> <221> variant &Lt; 222 > (44) <223> may be replaced by V or L <220> <221> variant &Lt; 222 > (46) .. (46) <223> may be replaced by A <220> <221> variant <222> (49). (49) <223> may be replaced by Q <220> <221> variant (52). (52) <223> may be replaced by D or S <220> <221> variant &Lt; 222 > (53) <223> may be replaced by E <220> <221> variant &Lt; 222 > (54) <223> may be replaced by S <220> <221> variant (58). (58) <223> may be replaced by K <400> 1 Ala Ala Ala Lys His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 2 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> Generic sequence 2 <220> <221> variant <222> (1) <223> may be replaced by N or P <220> <221> variant &Lt; 222 > (5) <223> may be replaced by F <220> <221> variant <222> (7) (7) <223> may be replaced by E <220> <221> variant &Lt; 222 > (8) <223> may be replaced by E ore <220> <221> variant &Lt; 222 > (11) <223> may be replaced by N <220> <221> variant &Lt; 222 > (25) <223> may be replaced by E <220> <221> variant &Lt; 222 > (28) <223> may be replaced by S <220> <221> variant &Lt; 222 > (44) <223> may be replaced by V <220> <221> variant &Lt; 222 > (46) .. (46) <223> may be replaced by A <220> <221> variant <222> (49). (49) <223> may be replaced by Q <220> <221> variant &Lt; 222 > (54) <223> may be replaced by S <220> <221> variant (58). (58) <223> may be replaced by K <400> 2 Ala Ala Ala Lys His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 3 <211> 58 <212> PRT <213> Artificial Sequence <220> Parental protein IB14 1A / 28N (IB14a) <400> 3 Ala Ala Ala Lys His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 4 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> parental protein IB27 1A <400> 4 Ala Ala Ala Lys Phe Asp Glu Ala Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Val Leu Gly Glu Ala         35 40 45 Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys     50 55 <210> 5 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> domain C <400> 5 Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys     50 55 <210> 6 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> domain B <400> 6 Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Ser Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys     50 55 <210> 7 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> domain Z <400> 7 Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Ser Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys     50 55 <210> 8 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> domain Z / 2 <400> 8 Val Asp Ala Lys Phe Asp Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Ser Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys     50 55 <210> 9 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> IB14 1I <400> 9 Ile Ala Ala Lys His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Ser Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 10 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> IB14 1A / 28S (IB14b) <400> 10 Ala Ala Ala Lys His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Ser Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 11 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> IB14 11A <400> 11 Pro Ala Ala Lys His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Ser Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 12 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> IB14 11E <400> 12 Pro Ala Ala Lys His Asp Lys Asp Gln Gln Glu Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Ser Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 13 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> IB14 11I <400> 13 Pro Ala Ala Lys His Asp Lys Asp Gln Gln Ile Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Ser Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 14 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> IB14 1P 28S (IB14d) <400> 14 Pro Ala Ala Lys His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Ser Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 15 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> IB14 35R <400> 15 Pro Ala Ala Lys His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Ser Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 16 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> IB14 35I <400> 16 Pro Ala Ala Lys His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Ser Ala Phe Ile Gln             20 25 30 Ser Leu Ile Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 17 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> IB14 42L <400> 17 Pro Ala Ala Lys His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Ser Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 18 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain cs14-1 (IB14 1I / 11A / 35R) <400> 18 Ile Ala Ala Lys His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Ser Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 19 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain cs14-2 (IB14 1I / 11A / 35R / 42L) <400> 19 Ile Ala Ala Lys His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Ser Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 20 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain cs14-3 1I / 11A / 28N / 35R / 42L <400> 20 Ile Ala Ala Lys His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 21 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> IB14 1P / 28N (IB14c) <400> 21 Pro Ala Ala Lys His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 22 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> IB27 1N <400> 22 Asn Ala Ala Lys Phe Asp Glu Ala Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Val Leu Gly Glu Ala         35 40 45 Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys     50 55 <210> 23 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> cs27 delNC <400> 23 Lys Phe Asp Glu Ala Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu 1 5 10 15 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg             20 25 30 Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu Ala Gln Lys Leu         35 40 45 Asn Asp Ser Gln Ala     50 <210> 24 <211> 55 <212> PRT <213> Artificial Sequence <220> <223> cs27 delN <400> 24 Ala Phe Asp Glu Ala Gln Ala Phe Tyr Glu Ile Leu His Leu 1 5 10 15 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg             20 25 30 Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu Ala Gln Lys Leu         35 40 45 Asn Asp Ser Gln Ala Pro Lys     50 55 <210> 25 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> parental protein IB25 1A <400> 25 Ala Ala Ala Lys His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys     50 55 <210> 26 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain cs25 (IB25 1I / 11A / 35R / 42L) <400> 26 Ile Ala Ala Lys His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Ala Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys     50 55 <210> 27 <211> 318 <212> PRT <213> Artificial Sequence <220> <223> cs17 delNC hexamer <400> 27 Lys Phe Asp Glu Ala Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu 1 5 10 15 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg             20 25 30 Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu Ala Gln Lys Leu         35 40 45 Asn Asp Ser Gln Ala Lys Phe Asp Glu Ala Gln Gln Ala Ala Phe Tyr     50 55 60 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 65 70 75 80 Ile Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly                 85 90 95 Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala Lys Phe Asp Glu Ala Gln             100 105 110 Gln Ala Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu         115 120 125 Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro Ser Val Ser     130 135 140 Leu Glu Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala Lys 145 150 155 160 Phe Asp Glu Ala Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu Pro                 165 170 175 Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp             180 185 190 Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu Ala Gln Lys Leu Asn         195 200 205 Asp Ser Gln Ala Lys Phe Asp Glu Ala Gln Gln Ala Ala Phe Tyr Glu     210 215 220 Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile 225 230 235 240 Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu                 245 250 255 Ala Gln Lys Leu Asn Asp Ser Gln Ala Lys Phe Asp Glu Ala Gln Gln             260 265 270 Ala Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln         275 280 285 Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu     290 295 300 Glu Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala 305 310 315 <210> 28 <211> 290 <212> PRT <213> Artificial Sequence <220> <223> cs27 pentamer <400> 28 Ile Ala Lys Phe Asp Glu Ala Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu Ala         35 40 45 Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys Ile Ala Ala Lys Phe Asp     50 55 60 Glu Ala Gln Ala Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu 65 70 75 80 Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro                 85 90 95 Ser Val Ser Leu Glu Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser             100 105 110 Gln Ala Pro Lys Ile Ala Ala Lys Phe Asp Glu Ala Gln Gln Ala Ala         115 120 125 Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn     130 135 140 Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val 145 150 155 160 Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys Ile Ala                 165 170 175 Ala Lys Phe Asp Glu Ala Glu Gln Ala Ala Phe Tyr Glu Ile Leu His             180 185 190 Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu         195 200 205 Arg Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu Ala Gln Lys     210 215 220 Leu Asn Asp Ser Gln Ala Pro Lys Ile Ala Ala Lys Phe Asp Glu Ala 225 230 235 240 Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu                 245 250 255 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro Ser Val             260 265 270 Ser Leu Glu Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala         275 280 285 Pro Lys     290 <210> 29 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain cs27-1 (IB27 1I / 11A / 35R) <400> 29 Ile Ala Lys Phe Asp Glu Ala Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Lys Glu Val Leu Gly Glu Ala         35 40 45 Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys     50 55 <210> 30 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain cs27-2 (IB27 I / 11A / 35R / 42L) <400> 30 Ile Ala Lys Phe Asp Glu Ala Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu Ala         35 40 45 Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys     50 55 <210> 31 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain C 1 I / 11 A / 35 R <400> 31 Ile Asp Asn Lys Phe Asn Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys     50 55 <210> 32 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain C 1 I / 11 A / 35 R / 42 L <400> 32 Ile Asp Asn Lys Phe Asn Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Ala Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys     50 55 <210> 33 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain B 1I / 11A / 35R <400> 33 Ile Asp Asn Lys Phe Asn Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Ser Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys     50 55 <210> 34 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain B 1I / 11A / 35R / 42L <400> 34 Ile Asp Asn Lys Phe Asn Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Gln Ser Leu Asn Leu Leu Ala Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys     50 55 <210> 35 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain Z 1I / 11A / 35R <400> 35 Ile Asp Asn Lys Phe Asn Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Ser Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys     50 55 <210> 36 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain Z 1I / 11A / 35R / 42L <400> 36 Ile Asp Asn Lys Phe Asn Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Gln Ser Leu Asn Leu Leu Ala Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys     50 55 <210> 37 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain domain Z / 2 1I / 11A / 35R <400> 37 Ile Asp Ala Lys Phe Asp Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Ser Ser Seru Ala Leu Leu Ala Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys     50 55 <210> 38 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain Z / 2 1I / 11A / 35R / 42L <400> 38 Ile Asp Ala Lys Phe Asp Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Gln Ser Leu Asn Leu Leu Ala Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys     50 55 <210> 39 <211> 79 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain cs14-3 with coupling sequence        and strep tag <400> 39 Ile Ala Ala Lys His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro Ala Ser Pro Ala Pro Ser     50 55 60 Ala Pro Ser Ala Cys Ala Ser Trp Ser His Pro Gln Phe Glu Lys 65 70 75 <210> 40 <211> 79 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain cs27-2 with coupling sequence        and strep tag <400> 40 Ile Ala Lys Phe Asp Glu Ala Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu Ala         35 40 45 Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Ser Pro Ala Pro Ser     50 55 60 Ala Pro Ser Ala Cys Ala Ser Trp Ser His Pro Gln Phe Glu Lys 65 70 75 <210> 41 <211> 13 <212> PRT <213> Artificial Sequence <220> Coupling sequence <400> 41 Ala Ser Pro Ala Pro Ser Ala Pro Ser Ala Cys Ala Ser 1 5 10 <210> 42 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain cs74h1 <400> 42 Ile Ala Lys Phe Asp Glu Ala Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 43 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain cs74h2 <400> 43 Ile Ala Lys Phe Asp Glu Ala Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 44 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain cs47H3 <400> 44 Ile Ala Ala Lys His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu Ala         35 40 45 Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys     50 55 <210> 45 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> alkaline stable Ig binding domain cs47H4 <400> 45 Ile Ala Ala Lys His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu Ala         35 40 45 Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys     50 55 <210> 46 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> StrepTag <400> 46 Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 47 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> parental protein IB74h1 <400> 47 Ala Ala Ala Lys Phe Asp Glu Ala Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 48 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> Parental protein IB74h2 <400> 48 Ala Ala Ala Lys Phe Asp Glu Ala Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 49 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> parental protein IB47h3 <400> 49 Ala Ala Ala Lys His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Val Leu Gly Glu Ala         35 40 45 Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys     50 55 <210> 50 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> parental protein IB47h4 <400> 50 Ala Ala Ala Lys His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Val Leu Gly Glu Ala         35 40 45 Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys     50 55 <210> 51 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> generic sequence including IB14 and IB27 <220> <221> variant <222> (1) <223> may be replaced by N or P <220> <221> variant &Lt; 222 > (5) <223> may be replaced by F <220> <221> variant <222> (7) (7) <223> may be replaced by E <220> <221> variant &Lt; 222 > (8) <223> may be replaced by A <220> <221> variant &Lt; 222 > (25) <223> may be replaced by E <220> <221> variant &Lt; 222 > (28) <223> may be replaced by S <220> <221> variant &Lt; 222 > (44) <223> may be replaced by V <220> <221> variant <222> (49). (49) <223> may be replaced by Q <220> <221> variant &Lt; 222 > (54) <223> may be replaced by S <220> <221> variant (58). (58) <223> may be replaced by K <400> 51 Ala Ala Ala Lys His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 52 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> Generic sequence of preferred alkaline stable artificial IgG        binding proteins <220> <221> variant &Lt; 222 > (5) <223> may be replaced by F <220> <221> variant <222> (7) (7) <223> may be replaced by E <220> <221> variant &Lt; 222 > (8) <223> may be replaced by A or E <220> <221> variant &Lt; 222 > (25) <223> may be replaced by E <220> <221> variant &Lt; 222 > (28) <223> may be replaced by S <220> <221> variant &Lt; 222 > (42) <223> may be replaced by L <220> <221> variant &Lt; 222 > (44) <223> may be replaced by V <220> <221> variant &Lt; 222 > (46) .. (46) <223> may be replaced by A <220> <221> variant <222> (49). (49) <223> may be replaced by Q <220> <221> variant &Lt; 222 > (54) <223> may be replaced by S <220> <221> variant (58). (58) <223> may be replaced by K <400> 52 Ile Ala Ala Lys His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 53 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> cs27 46C <400> 53 Ile Ala Lys Phe Asp Glu Ala Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val Leu Cys Glu Ala         35 40 45 Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys     50 55 <210> 54 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> cs14 43C <400> 54 Ile Ala Ala Lys His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Cys Ile Leu Gly Glu Ala         35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Pro     50 55 <210> 55 <211> 174 <212> PRT <213> Artificial Sequence <220> <223> cs27 tetramer <400> 55 Ile Ala Lys Phe Asp Glu Ala Gln Gln Ala Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln             20 25 30 Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val Leu Gly Glu Ala         35 40 45 Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys Ile Ala Ala Lys Phe Asp     50 55 60 Glu Ala Gln Ala Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu 65 70 75 80 Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro                 85 90 95 Ser Val Ser Leu Glu Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser             100 105 110 Gln Ala Pro Lys Ile Ala Ala Lys Phe Asp Glu Ala Gln Gln Ala Ala         115 120 125 Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn     130 135 140 Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Val 145 150 155 160 Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys                 165 170

Claims (22)

1. An immunoglobulin (Ig) binding protein comprising at least one Ig binding domain, wherein at least one Ig binding domain comprises an amino acid substitution at position 1 to isoleucine, alanine at position 11, glutamic acid, or The parent of SEQ ID NO: 1 having at least 1, 2, 3 or 4 substitutions selected from the group consisting of amino acid substitutions to isoleucine, amino acid substitutions to isoleucine, amino acid substitutions to arginine or isoleucine at position 35, and amino acid substitutions to leucine at position 42 An immunoglobulin (Ig) binding protein comprising a variant of an amino acid sequence. 1. An immunoglobulin (Ig) binding protein comprising at least one Ig binding domain, wherein at least one Ig binding domain comprises an amino acid substitution at position 1 to isoleucine, alanine at position 11, glutamic acid, or 2 having at least 1, 2, 3 or 4 substitutions selected from the group consisting of amino acid substitutions to isoleucine, amino acid substitutions to isoleucine, amino acid substitutions to arginine or isoleucine at position 35, and amino acid substitutions to leucine at position 42 An immunoglobulin (Ig) binding protein comprising a variant of an amino acid sequence. The Ig binding domain according to claim 1 or 2, wherein the Ig binding domain comprises an amino acid substitution of alanine or proline to isoleucine at position 1, an amino acid substitution of serine alanine, glutamic acid, or isoleucine at position 11, arginine or isoleucine of lysine at position 35 2, 3, or 4 amino acid substitutions selected from the group consisting of amino acid substitutions of lysine to leucine at position 42 and amino acid substitutions of leucine to leucine at position 42, and SEQ ID NOs: 3, 4, 10, 14, 21, 25, 47, 48, 49, and 50, respectively. The Ig binding domain of claim 1, wherein said Ig binding domain comprises a variant of a parental amino acid sequence of any one of SEQ ID NOS: 5-8, wherein said variant is an amino acid substitution at position 1 to isoleucine, at alanine, glutamic acid, or isoleucine 2, 3, or 4 amino acid substitutions selected from the group consisting of amino acid substitutions at position 35 to arginine or isoleucine, and amino acid substitutions at position 42 to leucine. . The method according to any one of claims 1 to 4, wherein the variant is selected from the group consisting of 1I; 11A; 35R; 42L; 11E; 11I; 35I; 11I / 11A; 1I / 35R; 11A / 35R; 1I / 42L; 11A / 42L; 11 / 11E; 11I / 11I; 11I / 35R; 11E / 35R; 11I / 42L; 11E / 42L; 1I / 35I; 11A / 35I; 11I / 35I; 11E / 35I; 35R / 42L; 35I / 42L; 1I / 11A / 35R; 1I / 11E / 35R; 1I / 11I / 35R; 1I / 11A / 42L; 1I / 11E / 42L; 1I / 11I / 42L; 1I / 11A / 35I; 1I / 11E / 35I; 1I / 11I / 35I; 1I / 35R / 42L; 1I / 35I / 42L; 11I / 35R / 42L; 11I / 35I / 42L; 11A / 35R / 42L; 11A / 35I / 42L; 11E / 35R / 42L; 11E / 35I / 42L; 1I / 11A / 35R / 42L; 1I / 11E / 35R / 42L; 1I / 11I / 35R / 42L; 1I / 11A / 35I / 42L; 1I / 11E / 35I / 42L; 1I / 11I / 35I / 42L; 1I / 11A / 28N / 35R / 42L; 1I / 11E / 28N / 35R / 42L; 1I / 11I / 28N / 35R / 42L; 1I / 11A / 28N / 35I / 42L; 1I / 11I / 28N / 35I / 42L; And 1I / 11E / 28N / 35I / 42L. 6. The Ig binding protein of claim 5, wherein 3 or 4 of the amino acid positions are selected from the group consisting of 1I, 11A, 35R, and 42L. 7. The method of any one of claims 1-6, wherein the Ig binding domain further comprises 1, 2, 3, 4, 5, or 6 variants, wherein each individual variation comprises a single amino acid substitution, &Lt; / RTI &gt; a single amino acid insertion. 52. An Ig binding protein comprising at least one Ig binding domain, wherein said at least one Ig binding domain comprises the amino acid sequence of SEQ ID NO: 52, wherein the amino acid at position 5 is selected from H or F, Is selected from K or E, the amino acid at position 8 is selected from D, A, or E, the amino acid at position 25 is selected from D or E, the amino acid at position 28 is selected from S or N, The amino acid is selected from L or K, preferably L, the amino acid at position 44 is selected from I or V, the amino acid at position 46 is selected from G or A, the amino acid at position 49 is selected from K or Q, An Ig binding protein wherein the amino acid at position 54 is selected from A or S, and the amino acid at position 58 is selected from P or K; 9. The method of claim 8, wherein the Ig binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 26, 30, and 42-45, or comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: Wherein the sequence is at least 89.5% sequence identity to the amino acid sequence selected from the group consisting of SEQ. The Ig binding protein according to any one of claims 1 to 9, wherein the Ig binding domain comprises the amino acid sequence set forth in SEQ ID NO: 20, or a sequence at least 96% identical thereto. The Ig binding protein of any one of claims 1 to 10, wherein the Ig binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 30, or a sequence at least 94% identical thereto. The Ig binding protein of any one of claims 1-11, wherein the Ig binding domain comprises the amino acid sequence set forth in SEQ ID NO: 26, or a sequence at least 98% identical thereto. The Ig binding protein of any one of claims 1 to 12, wherein the Ig binding domain comprises the amino acid sequence set forth in SEQ ID NO: 44, or a sequence at least 98% identical thereto. The Ig binding protein of any one of claims 1-13, wherein the Ig binding domain comprises the amino acid sequence set forth in SEQ ID NO: 43, or a sequence at least 98% identical thereto. The Ig binding protein according to any one of claims 1 to 14, wherein the protein comprises 2, 3, 4, 5, 6, 7, or 8 Ig binding domains linked to each other. The Ig binding protein according to any one of claims 1 to 15, wherein the protein is conjugated to a solid support. 17. The Ig binding protein of claim 16, wherein the Ig binding protein further comprises an attachment site for site-specific covalent coupling of the Ig binding protein to a solid support. The Ig binding protein according to any one of claims 1 to 17, wherein the Ig binding protein comprises an Ig fragment comprising IgG1, IgG2, IgG4, IgM, IgA, Fc region, a fusion protein comprising an Fc region in Ig, Lt; RTI ID = 0.0 &gt; Ig &lt; / RTI &gt; binding protein. An affinity separation matrix comprising an Ig binding protein as defined in any one of claims 1 to 18. Use of the Ig binding protein of any one of claims 1 to 18 or the affinity separation matrix of claim 19 for affinity purification of immunoglobulins. A method of purifying an affinity of an immunoglobulin, said method comprising: (a) providing a liquid containing an immunoglobulin; (b) providing an affinity separation matrix comprising at least one Ig binding protein of any one of claims 1 to 18 coupled to an affinity separation matrix; (c) contacting the liquid with the affinity separation matrix, wherein the immunoglobulin binds to the Ig binding protein; And (d) eluting the immunoglobulin from the matrix, thereby obtaining an eluate containing the immunoglobulin. 22. The method of claim 21, further comprising, between steps (c) and (d), washing the affinity matrix under conditions sufficient to remove some or all of the molecules that are non- Further comprising the steps of:

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